It would be difficult to overestimate the contribution of genetic manipulation to the study of any biological system, and it is an essential tool for the metabolic engineering of biosynthetic and substrate utilization pathways. This is particularly true for the archaea since, in spite of their environmental and industrial importance, coupled with their unique molecular features, much remains to be learned about their biology (Allers and Mevarech, 2005 Nat. Rev. Genet. 6:58-73). The marine hyperthermophilic anaerobe Pyrococcus furiosus is of special interest not only for its ability to grow optimally at 100° C. and the implications of this trait for its biology but also for industrial applications of its enzymes, as well as its capacity to produce hydrogen efficiently (Atomi, 2005 Curr. Opin. Chem. Biol. 9:166-173; Egorova and Antranikian, 2005 Curr. Opin. Microbiol. 8:649-655; Verhaart et al., 2010 Environ. Technol. 31:993-1003).
The development of genetic systems in the archaea, in general, presents many unique challenges given the extreme growth requirements of many of these organisms. To date, genetic systems of various levels of sophistication have been developed for representatives of all major groups of archaea, including halophiles, methanogens, thermoacidophiles, and hyperthermophiles (Allers and Mevarech, 2005 Nat. Rev. Genet. 6:58-73; Berkner and Lipps, 2008 Arch. Microbiol. 190:217-230; Rother and Metcalf, 2005 Curr. Opin. Microbiol. 8:745-751; Soppa, 2006 Microbiology 152:585-590; Tumbula and Whitman, 1999 Mol. Microbiol. 33:1-7; Wagner et al., 2009 Biochem. Soc. Trans. 37:97-101). A variety of transormation methods are being used, including electroporation, heat shock with or without CaCl2 treatment, phage-mediated transduction, spheroplast transformation, liposomes, and, very recently, even conjugation with Escherichia coli (Allers and Mevarech, 2005 Nat. Rev. Genet. 6:58-73; Dodsworth et al., 2010 Appl. Environ. Microbiol. 76:5644-5647). Transformation via natural competence has been reported in three archaeal species, in comparison to over 60 bacterial species that are known to exhibit this trait (Johnsborg et al., 2007 Res. Microbiol. 158:767-778; Sato et al., 2003 J. Bacteriol. 185:210-220). Two of them are the methanogens Methanococcus voltae PS (Bertani and Baresi, 1987 J. Bacteriol. 169:2730-2738; Patel et al., 1994 Appl. Environ. Microbiol. 60:903-907) and Methanobacterium thermoautotrophicum Marburg (Worrell et al., 1988 J. Bacteriol. 170:653-656); however, transformation frequencies were low, and there have been no follow-up studies regarding natural competence. The other is the hyperthermophile Thermococcus kodakarensis, which has an optimal growth temperature of 85° C. Its natural competence has enabled the development of genetic tools for targeted gene deletions, the use of shuttle vectors, and a reporter gene system (Santangelo et al., 2008 J. Bacteriol. 190:2244-2248; Santangelo et al., 2008 Appl. Environ. Microbiol. 74:3099-3104; Santangelo et al., 2010 Appl. Environ. Microbiol. 76:1044-1052; Sato et al., 2005 Appl. Environ. Microbiol. 71:3889-3899; Sato et al., 2003 J. Bacteriol. 185:210-220; Sato et al., 2004 J. Bacteriol. 186:5799-5807). In fact, T. kodakarensis was one of the first archaeal hyperthermophiles for which chromosomal manipulations were reported (Sato et al., 2003 J. Bacteriol. 185:210-220), along with Sulfolobus solfataricus, for which a transformation system with accompanying shuttle vectors had previously been established (Berkner and Lipps, 2008 Arch. Microbiol. 190:217-230; Worthington et al., 2003 J. Bacteriol. 185:482-488). Sulfolobus sp., Thermococcus kodakaraensis, and Pyrococcus furiosus are all transformed by linear DNA fragments (Deng et al., 2009 Extremophiles 13:735-46; Grogan and Stengel, 2008 Mol. Microbiol. 69:1255-1265; Kurosawa and Grogan, 2005 FEMS Microbiol. Lett. 253:141-9; Sato et al., 2003 J. Bacteriol. 185:210-220; Sato et al., 2005 Appl. Environ. Microbiol. 71:3889-3899; Lipscomb et al., 2011 Appl. Environ. Microbiol. 77:2232-8 (Example I)) and the limits of homology needed for marker replacement by linear DNA varies. In T. kodakaraensis more than 100 bp of homologous flanking region is required for homologous recombination (Sato et al., 2005 Appl. Environ. Microbiol. 71:3889-3899), and S. acidocaldarius 10-30 bp of homology is sufficient (Kurosawa and Grogan, 2005 FEMS Microbiol. Lett. 253:141-9).
One of the most significant barriers to genetic manipulation of archaea, in general, and hyperthermophiles, in particular, is the lack of selectable markers. Antibiotic selection strategies used in mesophilic bacteria are typically ineffective because the molecular machineries of archaea are not affected by the antibiotic (Cammarano et al., 1985 EMBO J. 4:811-816; Possot et al., 1988 Appl. Environ. Microbiol. 54:734-740) or, in the case of hyperthermophiles, because of the instability of either the drug or the heterologously expressed resistance protein at high temperatures (Allers and Mevarech, 2005 Nat. Rev. Genet. 6:58-73; Noll and Vargas, 1997 Arch. Microbiol. 168:73-80). One exception is the drug simvastatin (or mevinolin), first used in the haloarchaea (Lam and Doolittle, 1989 Proc. Natl. Acad. Sci. U.S.A. 86:5478-5482; Peck et al., 2000 Mol. Microbiol. 35:667-676), which is sufficiently thermostable to inhibit growth of both T. kodakarensis (85° C.) (Matsumi et al., 2007 J. Bacteriol. 189:2683-2691) and P. furiosus (Waege et al., 2010 Appl. Environ. Microbiol. 76:3308-3313). Simvastatin competitively inhibits 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, the rate-limiting step in the biosynthesis of isoprenoids, the major component of archaeal membrane lipids. Simvastatin at sufficiently high concentrations leads to cessation of cell growth, while overexpression of HMG-CoA reductase confers resistance (Lam and Doolittle, 1989 Proc. Natl. Acad. Sci. U.S.A. 86:5478-5482; Matsumi et al., 2007 J. Bacteriol. 189:2683-2691).
Nutritional markers are especially useful for genetic selection if an organism is able to grow on a defined medium, and a number of such nutritional selections have been used in archaea, including auxotrophies for amino acids (e.g., leucine and tryptophan) (Allers et al., 2004 Appl. Environ. Microbiol. 70:943-953; Sato et al., 2003 J. Bacteriol. 185:210-220), thymidine (Allers et al., 2004 Appl. Environ. Microbiol. 70:943-953; Ortenberg et al., 2000 Mol. Microbiol. 35:1493-1505), and agmatine (Santangelo et al., 2010 Appl. Environ. Microbiol. 76:1044-1052; Santangelo and Reeve, 2011, In: Extremophiles Handbook, Horikoshi (ed.), Springer, Chapter 4.8, pages 567-582). A counterselectable marker based on loss of the uracil biosynthetic enzyme orotidine-5′-monophosphate (OMP) decarboxylase, first described in yeast (Saccharomyces cerevisiae) (Boeke et al., 1984 Mol. Gen. Genet. 197:345-346), has been used successfully in archaeal organisms, including T. kodakarensis (Lucas et al., 2002 Appl. Environ. Microbiol. 68:5528-5536; Peck et al., 2000 Mol. Microbiol. 35:667-676; Sato et al., 2005 Appl. Environ. Microbiol. 71:3889-3899; Sato et al., 2003 J. Bacteriol. 185:210-220). OMP decarboxylase (pyrF in archaea and bacteria) converts the pyrimidine analog 5-fluoroorotic acid (5-FOA) to fluorodeoxyuridine, a toxic product that kills growing cells (Boeke et al., 1984 Mol. Gen. Genet. 197:345-346). Mutations in pyrF result in uracil auxotrophs that are resistant to 5-FOA.